CRISPR / Cas12a freeze-dried microsphere, three-microsphere detection system, application and kit

Abstract

The invention belongs to the field of biology, and discloses a CRISPR / Cas12a freeze-dried microsphere, which is characterized in that the CRISPR / Cas12a freeze-dried microsphere is obtained by freeze-drying the following raw materials: enzyme-free water, 10 * HOLMES Buffer for Cas12a, an RNA enzyme inhibitor, CRISPR / Cas12a protein, crRNA, an upstream primer F, a downstream primer R, a fluorescent probe Reporter and a freeze-drying protective additive; the CRISPR / Cas12a protein is an LbaCas12a protein, and the CRISPR / Cas12a protein is a Cas12a protein; the freeze-drying protective agent is prepared from mannitol, trehalose, polyethylene glycol, glucan and BSA (Bovine Serum Albumin). By further optimizing a freeze-drying protective agent, freeze-drying microballing of a CRISPR / Cas12a system is achieved, the freeze-drying microballing can be matched with RPA constant-temperature amplification double microballing, and detection is conducted through a one-step method. Meanwhile, the invention further discloses a three-microsphere detection system, application and a kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, and particularly relates to a CRISPR / Cas12a freeze-dried microsphere, a three-microsphere detection system, an application and a kit. BACKGROUND

[0002] The applicant previously filed an invention patent application with the publication number CN113234856A, the subject of which is a DENV one-step nucleic acid detection method based on CRISPR / Cas12a and isothermal amplification; the patent application places RT-RPA and CRISPR / Cas12a in the same reaction system to amplify and detect the nucleic acid to be detected, thereby achieving the purpose of one-step nucleic acid detection of DENV.

[0003] Meanwhile, in the scheme, the RPA reagent adopts freeze-dried RPA reaction microspheres.

[0004] In order to further improve the convenience of detection, it is a research direction to microsphere the CRISPR / Cas12a system as well;

[0005] In the field, the microsphere of the CRISPR / Cas12a system can be seen in the following technical solutions:

[0006] The patent application with the publication number CN119351627A, the subject of which is a detection method and application for sensitive detection of monkeypox virus at constant temperature;

[0007] The patent application with the publication number CN119320849A, the subject of which is a nucleic acid combination, kit and method for detecting monkeypox virus based on CRISPR technology;

[0008] The above two schemes respectively disclose the microsphere of the CRISPR / Cas12a system, and the freeze-drying protectants used are polyethylene glycol, trehalose, mannitol and polyethylene glycol, trehalose, sucrose, mannitol, respectively;

[0009] During the research of the present project, polyethylene glycol, trehalose, mannitol and polyethylene glycol, trehalose, sucrose, mannitol were used as freeze-drying protectants, and it was found that better one-step detection results could not be achieved. SUMMARY

[0010] The purpose of the present application is to provide a CRISPR / Cas12a freeze-dried microsphere, which realizes the freeze-drying microsphere of the CRISPR / Cas12a system through further optimization of the freeze-drying protectant, and the freeze-dried microsphere can cooperate with the RPA isothermal amplification double-microsphere to perform detection through one-step method.

[0011] Meanwhile, the present application also discloses a three-microsphere detection system, an application and a kit.

[0012] To achieve the above object, the application discloses the following:

[0013] A CRISPR / Cas12a lyophilized microsphere is obtained by lyophilization with enzyme-free water, 10×HOLMES Buffer for Cas12a, RNase inhibitor, CRISPR / Cas12a protein, crRNA, upstream primer F, downstream primer R, fluorescent probe Reporter, and lyoprotectant as raw materials;

[0014] The CRISPR / Cas12a protein is LbaCas12a protein.

[0015] The lyoprotectant is composed of mannitol, trehalose, polyethylene glycol, dextran, and BSA.

[0016] The preparation method of the CRISPR / Cas12a lyophilized microsphere comprises the following steps: adding the reaction liquid dropwise into liquid nitrogen to form a quick-frozen microsphere, placing the formed lyophilized microsphere at-80℃ for pre-freezing for 1-2 hours, and placing the pre-frozen reagent in a-80℃ environment for drying for 4-6 hours to obtain the lyophilized microsphere. Lyophilization is a process of drying the reaction mixture under ultra-low temperature conditions, and the purpose is to enable the reaction mixture to be stored and transported under normal temperature conditions, and to reduce the probability of enzyme activity reduction in long-distance transportation. The reaction mixture is divided into lyophilization containers, pre-frozen at-80℃ for 60-120 minutes, and then placed in a vacuum freeze dryer for drying, and the process is-80℃, 10-16 hours. The finished product after freeze-drying needs to be a white complete spherical ball, which can be clamped using forceps and the like, and after resuspension with water, it can be completely dissolved in 3-5 seconds without any granular residues.

[0017] In the above-mentioned CRISPR / Cas12a lyophilized microsphere, the weight ratio of the mannitol, trehalose, polyethylene glycol, dextran, and BSA is 1-5:1-8:2-4:1-5:1-5.

[0018] In the above-mentioned CRISPR / Cas12a lyophilized microsphere, the amount of the lyoprotectant in the raw materials is 6-22wt%.

[0019] In the above-mentioned CRISPR / Cas12a lyophilized microsphere, in the raw materials, the concentrations of the components are as follows: the final concentration of 10×HOLMES Buffer for Cas12a is 0.5×-2×; the concentration of the RNase inhibitor is 0.2-1U / μL; the concentration of the CRISPR / Cas12a protein is 10-100nm / μL; the concentration of the crRNA is 20-200nm / μL; the concentrations of the upstream primer F and the downstream primer R are respectively 200-300nM / μL; and the concentration of the fluorescent probe Reporter is 500-1000nM / μL.

[0020] Meanwhile, the application also discloses a three-microsphere detection system comprising the CRISPR / Cas12a freeze-dried microsphere and the RPA constant-temperature amplification double microsphere.

[0021] In the three-microsphere detection system, the RPA constant-temperature amplification double microsphere comprises an RPA freeze-dried microsphere and a Buffer freeze-dried microsphere; the RPA freeze-dried microsphere comprises a recombinase, a single-strand binding protein, a DNA polymerase, an auxiliary protein, polyethylene glycol, trehalose and mannitol; and the Buffer freeze-dried microsphere comprises Tris, potassium acetate, dithiothreitol, adenosine triphosphate, creatine phosphate disodium salt, creatine kinase and deoxyribonucleotide triphosphate.

[0022] The concentration ranges of the components of the reagent are as follows: the recombinase is 520-725 ng / μL; the single-strand DNA binding protein is 100-200 ng / μL; and the DNA polymerase is 350-450 ng / μL.

[0023] The Buffer freeze-dried microsphere is prepared by freeze-drying raw materials with the following concentrations: Tris, 20-40 mM; potassium acetate (Kac), 100-140 mM; dithiothreitol (DTT), 4-8 mM; adenosine triphosphate (ATP), 2-4 mM; creatine phosphate disodium salt (PCr), 40-60 mM; creatine kinase (CK), 80-120 ng / μl; and deoxyribonucleotide triphosphate (dNTP), 250-300 uM. These components provide the cofactors, salt ion concentrations, pH and reaction energy required for the reaction.

[0024] The buffer, protein and nucleic acid involved in the application are respectively the reaction buffer, the mouse RNAse inhibitor, the CRISPR / Cas12a nuclease, the ribonucleic acid required for the action of the CRISPR / Cas12a nuclease, the upstream and downstream primers required for the constant-temperature amplification process, the nucleic acid probe Reporter which is cut by the Cas12a enzyme to generate a fluorescent signal in the nucleic acid detection process, the recombinase, the single-strand binding protein, the DNA polymerase and the auxiliary protein which cooperate with each other to realize the rapid amplification of nucleic acid under the condition of constant temperature. They have the characteristics that the buffer components are prepared by optimizing the ion concentration and pH, and provide the most suitable working environment (pH 7.0-8.2, containing Mg 2+ / K +Plasma is used to ensure its catalytic activity and stability; proteins are derived from microorganisms, genetically engineered and expressed in microorganisms, and then extracted and purified. Their function is to target and cleave the target DNA sequence, generating a fluorescent signal during cleavage, and providing upstream and downstream primers for isothermal amplification. Buffers are from Tulu Harbour Biotechnology Co., Ltd.; mouse RNase inhibitors are derived from mice; Cas12a is derived from Lachnospiraceae bacterium; nucleic acids are obtained through chemical synthesis to obtain DNA or RNA sequences with specific sequences. Single-stranded DNA-binding proteins are derived from T4 or E. coli gp32; DNA polymerases have various sources, such as Phi-29 polymerase, or Bacillus subtilis Pol I (Bsu), Bst polymerase, or the large fragment of E. coli DNA polymerase I Klenow; accessory proteins are derived from T4 phage uvsY protein.

[0025] The sample liquid to be tested was added to a reaction tube containing Cas microspheres and RPA isothermal amplification dual microspheres, wherein the RPA isothermal amplification dual microspheres were sourced from Anpu Future (Changzhou) Biotechnology Co., Ltd. The reaction tube was placed in a temperature-controlled device and incubated at 20℃-42℃ for 30-75 minutes. This invention provides a method for detecting target nucleic acid fragments. Under isothermal conditions, the combined action of CRISPR / Cas12a nuclease, crRNA, specific primers, reporter probes, and components from the RPA isothermal amplification dual microspheres enables rapid on-site detection of target nucleic acid fragments using an instrument with fluorescence signal display or reading capabilities. This method avoids complex operational steps and can be transported, stored, and operated under ambient temperature conditions. It is also suitable for various fluorescence detection devices.

[0026] Ambient temperature and constant temperature conditions refer to conditions where precise temperature control equipment is not required, but only approximate temperature control is needed, such as water baths, metal baths, and incubators. Reaction temperatures are typically between 20℃ and 42℃. For the DNA targets to be detected, the optimal reaction temperature is 35℃ to 39℃.

[0027] Fluorescence detection equipment refers to devices that can simultaneously and in real time read fluorescence signals from multiple reaction tubes. It typically has a fluorescence detection channel and a heating module to maintain the reaction tubes under the required room temperature and isothermal conditions. It also refers to instruments that can place reaction tubes inside and convert the fluorescence value of the reaction system into an observable visible light signal through components such as light sources and filters.

[0028] The detection method of the present invention is suitable for application to a variety of fluorescence detection devices, and the results of the detection method can be interpreted by different types of fluorescence detection devices, including the devices of the above type.

[0029] Positive and negative determination of the sample to be detected: the mean value of the fluorescence value of the blank control group is used to calculate the standard deviation value of the sample, and the detection threshold of the sample is the mean value of the fluorescence detection of the blank control group plus three times the standard deviation value of the group (unit: fluorescence intensity). When the end fluorescence value of the sample is greater than the detection threshold, it is considered as positive amplification, otherwise it is considered as negative amplification.

[0030] The reaction tube of the present application is a closed tube detection. After adding the sample, the reaction tube is in a closed state, which can effectively avoid false positives caused by external aerosol pollution. At the same time, the present application is in the form of freeze-dried microspheres, which can be stored and transported under the above constant temperature conditions.

[0031] The principle of the detection method of the present application is: the target nucleic acid is exponentially enriched by RPA constant temperature amplification; the amplification product is specifically recognized by the Cas12a-crRNA complex, which activates the transcleavage activity of Cas12a; the activity non-specifically cleaves the fluorescent reporter probe (Reporter), dissociates the fluorescent group and the quencher group, and generates a detectable fluorescent signal; the fluorescence intensity is positively correlated with the target content.

[0032] In addition, the present application also discloses the use of the CRISPR / Cas12a freeze-dried microspheres as described above to prepare a detection kit.

[0033] In addition, the present application also discloses the use of the CRISPR / Cas12a freeze-dried microspheres as described above to prepare a detection kit.

[0034] Finally, the present application also discloses a detection kit containing the CRISPR / Cas12a freeze-dried microspheres as described above.

[0035] In addition, the present application also discloses a detection kit containing the CRISPR / Cas12a freeze-dried microspheres as described above.

[0036] The present application has at least the following beneficial effects:

[0037] 1) By using the freeze-dried microspheres together with the RPA system, the signal is amplified in cascade, the aM sensitivity of nucleic acid detection can be realized, and the specificity is high, and single base mutation can be detected.

[0038] 2) The thermal stability of the components in the system is improved by freeze-drying technology, and the system can be stored and transported at room temperature.

[0039] 3) By prepositioning the freeze-dried microspheres in the reaction tube, only the sample solution or standard solution needs to be added to the tube to realize nucleic acid detection, and the microspheres can be completely dissolved in 3-5s, which greatly simplifies the operation.

[0040] 4) The detection process does not require complex laboratory equipment, and the signal reading and result judgment can be performed by simple instruments. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Photo of Cas12 lyophilized microspheres 2;

[0042] Figure 2 Photo of Cas12 lyophilized microspheres 3;

[0043] Figure 3 Photo of Cas12 lyophilized microspheres 1, Cas12 lyophilized microspheres 4, Cas12 lyophilized microspheres 5;

[0044] Figure 4 Photo of single sphere test results;

[0045] Figure 5 Photo of Cas12 lyophilized microspheres 4, 5, three-microsphere detection system detection results;

[0046] Figure 6 Photo of Cas12 lyophilized microspheres 1, three-microsphere detection system detection results; DETAILED DESCRIPTION

[0047] The present application will be described in detail below with the embodiments of the present application. In the description of the present application, it should be noted that the specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are conventional products that can be purchased on the market. In the absence of special instructions, the parts used in the embodiments of the present application are all weight parts.

[0048] Example 1

[0049] Cas lyophilized microspheres of ASFV target

[0050] According to the genome of African swine fever virus, the crRNA of the target African swine fever virus and the upstream and downstream primers of the isothermal amplification were designed, the sequences are shown in Table 1, and the lyophilization system is shown in Table 2.

[0051] Table 1 Sequence list

[0052]

[0053] Table 2 Lyophilization reaction system

[0054] Component Working Concentration 10x HOLMES Buffer for Cas12a 1× RRI 1 U / μL Cas12a 100-150 nM crRNA 200-300 nM Primer_F 200 nM Primer_R 200 nM Reporter 500-1000 nM

[0055] The supplier information of each raw material is as follows:

[0056] 10×HOLMES Buffer for Cas12a: Tuoluangang Biotechnology Co., Ltd. (Part No. #32042);

[0057] RRI: Bio-Rad Laboratories, Inc. (Cat. No. JN5068);

[0058] RNP complex: Cas protein from TuRunPort Bio-Technology Co., Ltd. (Cat. No. V32108E), crRNA synthesized by Jinweizhi Bio-Technology Co., Ltd.;

[0059] Reporter: sequence F-TTATT-B, wherein F is a fluorescent group and B is a quenching group; the fluorescent group selected in this embodiment is 6-FAM; the quenching group selected in this embodiment is BHQ-1.

[0060] Based on Table 2 above, a freeze-drying protective agent was additionally added, and freeze-drying operation was performed;

[0061] The freeze-drying operation process was: pre-freezing at -80°C for 1.5 h, vacuum drying at -80°C for 12-16 h;

[0062] The formula of the freeze-drying protective agent is shown in Table 3;

[0063] Table 3 Freeze-drying protective agent Unit: w / v

[0064] Mannitol Polyethylene Glycol Trehalose Dextran Sucrose Lactose BSA Lyoprotectant 1 2％ 3％ 5％ 2％ 0 0 3％ Lyoprotectant 2 3％ 3％ 3％ 0 0 0 3％ Lyoprotectant 3 2％ 3％ 5％ 0 2％ 0 0 Lyoprotectant 4 2％ 3％ 5％ 0 0 2％ 0 Lyoprotectant 5 3％ 3％ 3％ 2％ 0 0 0

[0065] Polyethylene glycol is PEG-20000.

[0066] The above different freeze-drying protective agents and the freeze-drying reaction system shown in Table 2 prepared Cas12 freeze-dried microspheres 1, Cas12 freeze-dried microspheres 2, Cas12 freeze-dried microspheres 3, Cas12 freeze-dried microspheres 4, Cas12 freeze-dried microspheres 5; the number of freeze-dried microspheres corresponds to the number of freeze-drying protective agents.

[0067] After the above freeze-drying operation, Cas12 freeze-dried microspheres 2 and Cas12 freeze-dried microspheres 3 showed collapse phenomenon, referring to Figure 1 and Figure 2 ;

[0068] Cas12 freeze-dried microspheres 1, Cas12 freeze-dried microspheres 4, and Cas12 freeze-dried microspheres 5 have better forming degree, referring to Figure 3 ;

[0069] The Cas12 freeze-dried microspheres 1 were subjected to single sphere detection, and the detection method was:

[0070] According to the genome sequence of African swine fever virus published by genebank, the fragment was cloned into pUC57 plasmid as a detection target. It was diluted by gradient 10 times as a detection target to verify the detection performance of the microsphere product.

[0071] The Cas12 microspheres were placed in an eight-tube pipe, 3 μL of different concentrations of plasmid solution was diluted in 22 μL of enzyme-free water, and then added to the reaction tube, incubated at 37°C for 60 min.

[0072] The results are shown in Figure 4

[0073] The results show that: in the case of using Cas12 freeze-dried microspheres 1 alone, the target plasmid with a concentration of 10 -9 mol / L can be detected, and the fluorescence value of the blank control group remains at a low level.

[0074] Example 2

[0075] In this embodiment, different Cas freeze-dried microspheres and RPA constant-temperature amplification double-microsphere are used in combination, and the detection method is:

[0076] Three kinds of microspheres are assembled in the same tube, different concentrations of targets are added to the tube, and the reaction is carried out at 37°C for 1 h, and the fluorescence value is read by fluorescence quantification.

[0077] The supplier of RPA constant-temperature amplification double-microsphere is Anpu Future (Changzhou) Biotechnology Co., Ltd., and the product model is: basic type (customized);

[0078] The freeze-drying reaction system of Cas12 freeze-dried microspheres can be seen in Table 4, and the formula of the freeze-drying protectant can be seen in Table 3;

[0079] The detection limit of the three-microsphere detection system using different Cas12 freeze-dried microspheres can be seen in the following Table 4, and the Figure 5-6

[0080] Table 4 Detection limit of single-tube three-microsphere

[0081] Target plasmid detection limit Cas12 lyophilized microsphere 1 10 -16 mol / L Cas12 lyophilized microsphere 4 10 -13 mol / L Cas12 lyophilized microsphere 5 10 -15 mol / L

[0082] The results show that: using Cas12 freeze-dried microspheres 1 can obtain the lowest detection limit, and the fluorescence value of the blank control group remains at a low level.

[0083] The detection limit of Cas12 freeze-dried microspheres 4 and Cas12 freeze-dried microspheres 5 is lower than that of Cas12 freeze-dried microspheres 1, and the detected fluorescence value is lower;

[0084] From the above detection results, it can be known that although both lactose and dextran can keep Cas12 freeze-dried microspheres in an intact state, in terms of detection limit, it needs to be in the presence of PBS, and the selected polysaccharide is dextran.

[0085] It is illustrated that the selection and reasonable use of freeze-drying protectants are key factors to realize one-step detection.

[0086] ​​In summary, the application can quickly, real-time and specifically detect African swine fever virus, and through specific primers, fluorescent probes, CRISPR / Cas proteins and RPA constant-temperature amplification components, the rapid on-site nucleic acid detection of target nucleic acid molecules can be realized in an instrument with a fluorescence detection function. Meanwhile, nucleic acid detection can be realized through single sample addition, meeting the demand of simple operation for on-site nucleic acid detection. In addition, by replacing crRNA and constant-temperature amplification primer pairs in the freeze-drying system, the Cas microspheres can be used for detecting other different nucleic acid sequences.

Claims

1. A CRISPR / Cas12a lyophilized microsphere, characterized in that, The CRISPR / Cas12a lyophilized microspheres are prepared by using the following raw materials: enzyme-free water, 10x HOLMES Buffer for Cas12a, RNase inhibitor, CRISPR / Cas12a protein, crRNA, upstream primer F, downstream primer R, fluorescent probe Reporter, and lyoprotectant. The CRISPR / Cas12a protein is LbaCas12a protein. The lyoprotectant is composed of mannitol, trehalose, polyethylene glycol, dextran, and BSA.

2. The CRISPR / Cas12a lyophilized microsphere of claim 1, wherein, The weight ratio of the mannitol, trehalose, polyethylene glycol, dextran, and BSA is 1-5:1-8:2-4:1-5:1-5.

3. The CRISPR / Cas12a lyophilized microsphere of claim 1, wherein, The amount of the lyoprotectant in the raw materials is 6-22wt%.

4. The CRISPR / Cas12a lyophilized microsphere of claim 1, wherein, In the raw materials, the concentrations of the components are as follows: the final concentration of 10x HOLMES Buffer for Cas12a is 0.5x-2x; the concentration of RNase inhibitor is 0.2-1U / μL; the concentration of CRISPR / Cas12a protein is 10-100nm / μL; the concentration of crRNA is 20-200nm / μL; the concentrations of the upstream primer F and the downstream primer R are 200-300nM / μL respectively; and the concentration of the fluorescent probe Reporter is 500-1000nM / μL.

5. A tri-microsphere detection system, characterized by, The three-microsphere detection system comprises the CRISPR / Cas12a lyophilized microspheres and RPA constant-temperature amplification double-microspheres.

6. The tri-microsphere detection system of claim 5, wherein, The RPA constant-temperature amplification double-microspheres comprise RPA lyophilized microspheres and Buffer lyophilized microspheres; the RPA lyophilized microspheres comprise recombinase, single-strand binding protein, DNA polymerase, auxiliary protein, polyethylene glycol, trehalose, and mannitol; and the Buffer lyophilized microspheres comprise Tris, potassium acetate, dithiothreitol, adenosine 5'-phosphosulfate, phosphocreatine disodium salt, phosphocreatine kinase, deoxyribonucleoside triphosphate, and polyethylene glycol.

7. Use of the CRISPR / Cas12a lyophilized microspheres according to any one of claims 1-4 for preparing a detection kit.

8. Use of the three-microsphere detection system according to claim 5 or 6 for preparing a detection kit.

9. A test kit comprising, The detection kit contains the CRISPR / Cas12a lyophilized microspheres according to any one of claims 1-4.

10. A test kit comprising, The detection kit contains the three-microsphere detection system according to claim 5 or 6.

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